Quality assessment of circulating cell-free DNA using multiplexed droplet digital PCR

ABSTRACT

The present invention provides a method of determining integrity and/or quantity of cell free DNA (cfDNA) in a bio logical sample comprising amplifying target sequences with at least a first primer/probe set and at least a second primer probe/set, amplifying the target sequences of differing lengths, and monitoring for detection of the labels of the oligonucleotide probes, and determining the integrity and/or quantity of the cfDNA based on the level of detection of the label of the oligonucleotide probe from the first primer/probe set compared to the level detection of the label of the oligonucleotide probe from the second primer/probe set. The present invention also provides methods for generating a library with the cfDNA for sequencing and analysis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stake of International ApplicationNo. PCT/US2016/028159 filed Apr. 18, 2016, which claims priority to andthe benefit of U.S. Provisional Application No. 62/149,386 filed Apr.17, 2015, the contents of each of which are hereby incorporated byreference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII-formatted sequence listing with a file named“91482_179_Seq_Listing_ST25.txt” created on Apr. 14, 2016, and having asize of 6 kilobytes, and is filed concurrently with the specification.The sequence listing contained in this ASCII-formatted document is partof the specification and is herein incorporated by reference in itsentirety.

TECHNICAL FIELD

The present invention is related to methods of assessing the integrityand quantity of cell-free DNA in biological samples.

BACKGROUND

Analysis of circulating cell-free DNA (cfDNA) in plasma has severalestablished and upcoming diagnostic applications in prenatal, cancer andtransplant medicine (1-7). Whole-genome sequencing of cfDNA isclinically available for noninvasive prenatal screening of fetalaneuploidies (1, 5). Next-generation sequencing of cfDNA in cancerpatients can enable noninvasive identification of cancer mutations,monitoring of cancer burden and tumor evolution (4, 8-10). However,little is understood about the effect of pre-analytical factors on DNAquality and on performance of molecular assays.

Informative fraction of cfDNA is generally fragmented with a modal sizeof 160-180 bp. Pre-analytical factors such as delayed fractionation ofplasma or incomplete removal of peripheral blood cells before freezingcan cause an increase in higher molecular weight (HMW) DNA fraction fromcell lysis. For PCR-based sequencing approaches, this can artificiallylower the fraction of the target alleles such as a tumor-specificmutations in circulating tumor DNA making its detection more challengingand causing errors in its quantification. Since cfDNA is fragmented invivo, ligation-mediated preparation of sequencing libraries from cfDNAdoes not involve shearing, and therefore HMW DNA is not incorporatedinto sequencing libraries. If the upfront measurement of total cfDNA iserroneously high due to a large fraction of HMW DNA downstreamsequencing can be compromised.

Quantification of cfDNA can be performed using fluorometric orspectrophotometric methods such as QUBIT® (Life Technologies) orNANODROP™ (Thermo Scientific). These methods do not measure DNA size andcannot account for a HMW fraction in plasma DNA. Electrophoretic methodscan perform size-based quantification but require input amounts forreliable results that are not feasible for cfDNA analysis. In addition,none of these methods provide an assessment of amplifiable DNA copiesavailable for downstream molecular analysis. Multiplexed quantitativePCR can provide an assessment of size and amplifiable DNA but requirescomparison with a standard curve. It relies on 1-2 genomic loci to infertotal cfDNA content assuming that the targeted loci are single-copygenes. This assumption can affect measurement of cfDNA from cancerpatients with somatic copy number changes reflected in plasma. Inaddition, it was recently shown that relative readouts from multiplesingle-locus assays can vary systematically potentially due to assayperformance and variable stability across genomic DNA (11)

An efficient method for accurate quantification of cfDNA and assessmentof cfDNA integrity is needed. As the amounts of cfDNA available in asample are generally very limited, such a method requires a relativelywide dynamic range and the capability to detect and assess minuteamounts of cfDNA.

SUMMARY

The present invention is directed to a method of determining integrityand/or quantity of cell free DNA (cfDNA) in a biological sample, themethod comprising the steps of: a) obtaining a biological samplecomprising cfDNA; b) contacting the biological sample with at least afirst primer/probe set and at least a second primer probe/set, each ofwhich comprises at least one oligonucleotide probe that is detectablylabeled and at least two oligonucleotide primers and the labels aredifferent for each primer/probe set, under conditions such that thefirst primer/probe set anneals to a first target sequence of less than300 bp in length and the second primer/probe set anneals to a secondtarget sequence of 300 bp or greater in length; c) amplifying, ifpresent, the first target sequence and the second target sequence; andd) monitoring for detection of the label of the oligonucleotide probefrom the first primer/probe set as an indication of hybridization to thefirst target sequence and detection of the label of the oligonucleotideprobe from the second primer/probe set as an indication of hybridizationto the second target sequence; and e) determining the integrity and/orquantity of the cfDNA based on the level of detection of the label ofthe oligonucleotide probe from the first primer/probe set compared tothe level detection of the label of the oligonucleotide probe from thesecond primer/probe set, wherein a greater level of detection of thelabel of the oligonucleotide probe from the first primer/probe setcompared to the level of detection of the label of the oligonucleotideprobe from the second primer/probe set indicates increased integrityand/or quantity of cfDNA in the biological sample.

In some aspect, the oligonucleotide primers have a nucleotide sequencelength of about 10 to about 150. In other aspects, the oligonucleotideprobes have a nucleotide sequence length of about 10 to about 50.

In certain embodiments, the first target sequence and the second targetsequence are from at least one housekeeping gene. In one embodiment, theat least one housekeeping gene is selected from the group consisting ofACTB (Beta-actin), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase),RPLP0 (60 S acidic ribosomal protein P0), GUSB (beta-glucuronidase), andTFRC (transferring receptor 1).

In some aspects, the first primer/probe set comprises at least oneoligonucleotide probe and at least two oligonucleotide primers eachcomprising an oligonucleotide sequence selected from the groupconsisting of SEQ ID NOs: 13-27.

In other aspects, the second primer/probe set comprises at least oneoligonucleotide probe and at least two oligonucleotide primers eachcomprising an oligonucleotide sequence selected from the groupconsisting of SEQ ID NOs: 1-12.

In one embodiment, the first primer/probe set comprises at least two, atleast three, at least four, or at least five oligonucleotide probes thatare detectably labeled and at least four, at least six, at least eight,or at least 10 oligonucleotide primers, each of which anneals to atarget sequence of less than 300 bp.

In another embodiment, the second primer/probe set comprises at leasttwo, at least three, at least four, or at least five oligonucleotideprobes that are detectably labeled and at least four, at least six, atleast eight, or at least 10 oligonucleotide primers, each of whichanneals to a target sequence of 300 bp or greater in length.

In some aspects, detection of the labels of the oligonucleotide probesfrom the first primer probe/set and/or second primer/probe set areaveraged across each primer/probe set to determine the detection of thelabels.

In yet other aspects, the amplifying of the target sequences occurs witha digital PCR technique selected from droplet digital PCR (ddPCR),BEAMing (beads, emulsion, amplification, and magnetic), and microfluidicchips. In a particular embodiment, the digital PCR technique ismultiplexed ddPCR.

In one aspect, amplifiable copies (ACs) of cfDNA in the biologicalsample are estimated by: (i) dividing the number of positive dropletsindicating hybridization to the first target sequence by the number ofoligonucleotide probes in the first primer/probe set; (ii) dividing thenumber of positive droplets indicating hybridization to the secondtarget sequence by the number of oligonucleotide probes in the secondprimer/probe set; and (iii) subtracting (ii) from (i) to estimate ACs ofcfDNA in the biological sample.

In certain aspects, the oligonucleotide probes are detectably labeledwith a fluorescent label selected from the group consisting of FAM™ (5-or 6-carboxyfluorescein), Fluorescein, TET™ (5-tetrachloro-fluorescein),MARINA BLUE® (6,8-difluoro-7-hydroxy-4-methylcoumarin), ALEXA FLUOR® 350(7-amino-4-methyl-6-sulfocoumarin-3-acetic acid), YAKIMA YELLOW®(2′,5,5′,6-tetrachloro-7′-{12-[di(propan-2-yl)amino]-15-hydroxy-3-oxo-11,13-dioxa-4-aza-12-phosphapentadecyl}-4′-methyl-3-oxo-3H-spiro[2-benzofuran-1,9′-xanthene]-3′,6′-diylbis(2,2-dimethylpropanoate), and TEXAS RED® (sulforhodamine sulfonylchloride).

In one embodiment, the fluorescent labels are FAM™ (5- or6-carboxyfluorescein) and TET™ (5-tetrachloro-fluorescein).

In yet another embodiment, the first primer/probe set detects at leastone, at least two, at least three, at least four, or at least fivetarget sequences of about 50 bp to about 100 bp in length. In oneaspect, the second primer/probe set detects at least one, at least two,at least three, at least four, or at least five target sequences ofabout 300 bp to about 1000 bp in length.

In some aspects, the method further comprises generating a library withthe cfDNA for sequencing and analysis. In one embodiment, the library isan exome library.

In other aspects, the biological sample is divided into aliquots andeach aliquot undergoes a different upstream process to evaluate howupstream processing affects the integrity and/or quantity of cfDNA inthe biological sample.

In some embodiments, biological samples from different study cohorts areevaluated to determine the integrity and/or quality of the cfDNA in thebiological samples from each cohort.

In yet other embodiments, the biological sample is a biofluid selectedfrom the group consisting of blood, plasma, serum, saliva, urine, tears,and cerebral spinal fluid.

In one aspect, detection of the labels of the oligonucleotide probesoccurs with a flow cytometer or a droplet reader.

In yet another aspect, the method further comprises performing anamplification with the first primer/probe set and/or the secondprimer/probe set without the biological sample as a negative control.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows primer and probe sequences that can be used to quantify andassess the integrity of cfDNA. The lengths of the primers and probes,their melting temperatures (“Tm”), and GC content (“GC percent”) arealso indicated along with the length of the resulting amplicons(“Amplicon bp”).

FIG. 2 depicts the steps involved in one form of droplet digital PCR(ddPCR). In this figure, the sample contains a mixture of genomic DNAwith a mutant gene and genomic DNA with the corresponding wild typegene. The ddPCR is designed to amplify and detect relative amounts ofeach form of the gene with specific fluorescent probes.

FIG. 3 presents an embodiment of the ddPCR assay design which producesfive short amplicons (˜71 bp in length) and four long amplicons (˜471 bpin length). The nine genomic regions represent housekeeping regions. Thefluorescent probes shown in the figure comprise quenchers indicated witha “Q” and the fluorophores fluorescein amidite (FAM) and5-tetrachloro-fluorescein (TET), but any fluorophore known in the artcan be conjugated to the probes used in the assay.

FIG. 4 depicts a sample calculation of the number of amplifiable copiesof low molecular weight (LMW) cfDNA in a hypothetical biological samplecontaining 50 haploid copies of cfDNA with an average fragment size ofabout 180 bp and 20 haploid copies of DNA from intact peripheral bloodcells. The calculation is based on the ddPCR assay design shown in FIG.3.

FIG. 5 shows fluorescence measurements for large amplicons and shortamplicons with the ddPCR assay disclosed herein.

FIG. 6 depicts raw fluorescence intensity measurements from ampliconsgenerated with fragmented DNA.

FIG. 7 depicts genome equivalents per microliter of sample for intactgenomic DNA and fragmented genomic DNA having target fragment sizes of1000 bp, 500 bp, 300 bp, and 150 bp as calculated from the fluorescenceintensity measurements.

FIG. 8 shows actual curves (solid lines) generated with experimentaldata and simulated curves (dashed lines) based on exponentialdistributions with rates corresponding to average fragment sizes. ddPCRamplifiable copies at input fragment sizes of 150 bp, 300 bp, 500 bp,1,000 bp, and 10,000 bp are indicated in each curve.

FIG. 9 depicts the sequencing library yield for sheared vs. intact DNAwith intact DNA or sheared DNA comprising 25%, 50%, or 75% DNA fragmentsof about 300 bp.

FIG. 10A shows the fluorescence generated from two biological sampleswith differing amounts of HMW and LMW DNA using the multiplexed ddPCRassay. FIG. 10B depicts a bar graph indicating the relative amounts oflarge amplicons and small amplicons produced from each sample with themultiplexed ddPCR assay.

FIG. 11 depicts sequencing library diversity and size in relationship toddPCR optimized cfDNA input.

FIG. 12 presents the effect of several blood collection protocols on thenumber of cfDNA amplifiable copies per mL of plasma.

FIGS. 13A and 13B show that impact of several DNA extraction kits on theamount of cfDNA amplifiable copies obtained from plasma samples.Extraction kits identified as A, B, and C utilize spin columns whileextraction kits identified as D, E, F, G, and H utilize magnetic beads.

FIG. 14 depicts an analysis of the quality of biological samples fromdifferent study cohorts using the multiplexed ddPCR assay.

DETAILED DESCRIPTION

As used herein, the verb “comprise” as is used in this description andin the claims and its conjugations are used in its non-limiting sense tomean that items following the word are included, but items notspecifically mentioned are not excluded. In addition, reference to anelement by the indefinite article “a” or “an” does not exclude thepossibility that more than one of the elements are present, unless thecontext clearly requires that there is one and only one of the elements.The indefinite article “a” or “an” thus usually means “at least one.”

As used herein, “primer/probe set” refers to a grouping of a pair ofoligonucleotide primers and an oligonucleotide probe that hybridize to aspecific nucleotide sequence. Said oligonucleotide set consists of: (a)a forward discriminatory primer that hybridizes to a first location of anucleic acid sequence; (b) a reverse discriminatory primer thathybridizes to a second location of the nucleic acid sequence downstreamof the first location and (c) a fluorescent probe labeled with afluorophore and a quencher, which hybridizes to a location of thenucleic acid sequence between the primers. In other words, aprimer/probe set consists of a set of specific PCR primers capable ofinitiating synthesis of an amplicon specific to a nucleic acid sequence,and a fluorescent probe which hybridizes to the amplicon.

An “amplicon” refers to a nucleic acid fragment formed as a product ofnatural or artificial amplification events or techniques. For example,an amplicon can be produced by PCR, ligase chain reaction, or geneduplication.

The terms “short amplicon” and “small amplicon” used herein aresynonymous and refer to amplicons having a length less than 300 bp.

The terms “long amplicon” and “large amplicon” used herein aresynonymous and refer to amplicons having a length greater than or equalto 300 bp.

A “probe” or “fluorogenic probe” comprises an oligonucleotide sequencelabeled with both a “fluorescent reporter dye”, or “fluorophore”, and a“quencher dye”, or “quencher.” A “fluorescent reporter dye” or“fluorophore” refers to a molecule that emits light of a certainwavelength after having first absorbed light of a specific, but shorter,wavelength, wherein the emission wavelength is always higher than theabsorption wavelength. A “quencher dye” “quencher” refers to a moleculethat accepts energy from a fluorophore in the form of light at aparticular wavelength and dissipates this energy either in the form ofheat (e.g., proximal quenching) or light of a higher wavelength thanemitted from the fluorophore (e.g., FRET quenching). Quenchers generallyhave a quenching capacity throughout their absorption spectrum, but theyperform best close to their absorption maximum. For example, Deep DarkQuencher II absorbs over a large range of the visible spectrum and,consequently, efficiently quenches most of the commonly usedfluorophores, especially those emitting at higher wavelengths (like theCy® dyes). Similarly, the Black Hole Quencher family covers a largerange of wavelengths (over the entire visible spectrum and into thenear-IR). In contrast, Deep Dark Quencher I and Eclipse® Dark Quenchereffectively quench the lower wavelength dyes, such as FAM, but do notquench very effectively those dyes that emit at high wavelengths.

The term “housekeeping genes” as used herein is meant to refer to genesthat encode protein products that are not connected to, involved in orrequired for processes specific to a disease state (e.g., cancer) incells, and thus, exhibit a fixed expression level in diseased andhealthy cells. Examples of suitable housekeeping genes include, but arenot limited to, genes encoding ACTB (Beta-actin), GAPDH (Glyceraldehyde3-phosphate dehydrogenase), RPLP0 (60 S acidic ribosomal protein P0),GUSB (beta-glucuronidase), and TFRC (transferring receptor 1).

As used herein, the term “subject” or “patient” refers to any vertebrateincluding, without limitation, humans and other primates (e.g.,chimpanzees and other apes and monkey species), farm animals (e.g.,cattle, sheep, pigs, goats and horses), domestic mammals (e.g., dogs andcats), laboratory animals (e.g., rodents such as mice, rats, and guineapigs), and birds (e.g., domestic, wild and game birds such as chickens,turkeys and other gallinaceous birds, ducks, geese, and the like). Insome implementations, the subject may be a mammal, preferably a human.

As used herein, “digital PCR” refers to an assay that provides anend-point measurement that provides the ability to quantify nucleicacids without the use of standard curves, as is used in real-time PCR.In a typical digital PCR experiment, the sample is randomly distributedinto discrete partitions, such that some contain no nucleic acidtemplate and others contain one or more template copies. The partitionsare amplified to the terminal plateau phase of PCR (or end-point) andthen read to determine the fraction of positive partitions. If thepartitions are of uniform volume, the number of target DNA moleculespresent may be calculated from the fraction of positive end-pointreactions using Poisson statistics, according to the following equation:λ=−ln(1−p)  (1)wherein λ, is the average number of target DNA molecules per replicatereaction and p is the fraction of positive end-point reactions. From λ,together with the volume of each replicate PCR and the total number ofreplicates analyzed, an estimate of the absolute target DNAconcentration is calculated. Digital PCR includes a variety of formats,including droplet digital PCR, BEAMing (beads, emulsion, amplification,and magnetic), and microfluidic chips.

“Droplet digital PCR” (ddPCR) refers to a digital PCR assay thatmeasures absolute quantities by counting nucleic acid moleculesencapsulated in discrete, volumetrically defined, water-in-oil dropletpartitions that support PCR amplification (Hinson et al., 2011, Anal.Chem. 83:8604-8610; Pinheiro et al., 2012, Anal. Chem. 84:1003-1011). Asingle ddPCR reaction may be comprised of at least 20,000 partitioneddroplets per well.

A “droplet” or “water-in-oil droplet” refers to an individual partitionof the droplet digital PCR assay. A droplet supports PCR amplificationof template molecule(s) using homogenous assay chemistries and workflowssimilar to those widely used for real-time PCR applications (Hinson etal., 2011, Anal. Chem. 83:8604-8610; Pinheiro et al., 2012, Anal. Chem.84:1003-1011).

Droplet digital PCR may be performed using any platform that performs adigital PCR assay that measures absolute quantities by counting nucleicacid molecules encapsulated in discrete, volumetrically defined,water-in-oil droplet partitions that support PCR amplification. Thestrategy for droplet digital PCR may be summarized as follows: a sampleis diluted and partitioned into thousands to millions of separatereaction chambers (water-in-oil droplets) so that each contains one orno copies of the nucleic acid molecule of interest. The number of“positive” droplets detected, which contain the target amplicon (i.e.,nucleic acid molecule of interest), versus the number of “negative”droplets, which do not contain the target amplicon (i.e., nucleic acidmolecule of interest), may be used to determine the number of copies ofthe nucleic acid molecule of interest that were in the original sample.Examples of droplet digital PCR systems include the QX100™ DropletDigital PCR System by Bio-Rad, which partitions samples containingnucleic acid template into 20,000 nanoliter-sized droplets; and theRainDrop™ digital PCR system by RainDance, which partitions samplescontaining nucleic acid template into 1,000,000 to 10,000,000picoliter-sized droplets.

In some aspects, the present invention provides a method to performone-step quantification of DNA and assessment of DNA integrity(measurement of BMW and cfDNA fractions) using a 9-plex multiplexedapproach using ddPCR. Instead of measuring individual loci, the assayrelies on the average readout of all tested loci, measuring theconcentration of cfDNA and HMW DNA with high precision and accuracy.cfDNA quantification using this approach predicts downstream performanceof ligation-mediated exome sequencing.

In other aspects, the present invention provides an approach foraccurate one-step assessment of DNA quantity and integrity from minuteamounts of cell-free DNA using picoliter droplet digital PCR (ddPCR).

Ins some embodiments, a multiplexed ddPCR assay including primers andsequence-specific oligonucleotide probes to target 5 short amplicons(67-71 bp) and 4 long amplicons (439-522 bp) from independent quiescentregions of the human genome is provided. In one embodiment, all shortand long amplicon probes are labeled with fluorescent dyes FAM and TET,respectively. Amplifiable DNA fragments using short and long ampliconsare calculated as the average across each set, to increase precision andaccuracy when evaluating small input amounts of DNA. The assayperformance may be evaluated using control genomic DNA sheared bysonication to average fragment sizes of 150-1000 bp. 15 ligation-basedexome sequencing libraries from control plasma DNA samples, guided byddPCR DNA integrity assessment without further shearing were prepared tovalidate the assay.

In some aspects, relative quantities of amplifiable fragments usingshort and long PCR reflect integrity of sheared genomic DNA,approximately following expectations of the exponential distribution forDNA fragmentation. Accurate quantification and assessment of integrityis achievable using picogram amounts of cell-free DNA (as few as 200pg). DNA integrity and quantity assessments are predictive of librarydiversity and obtainable depth-of-coverage in next-generation sequencinglibraries made from cell-free DNA samples.

As novel diagnostic applications of circulating cell-free DNA areevaluated for clinical relevance, reliable assays for assessment of DNAquality are needed to objectively account for pre-analytical variation.The present invention provides an accurate and precise droplet digitalPCR approach to practically implement quality assurance for clinicalcell-free DNA studies.

In certain aspects, the present invention is directed to a method ofdetermining integrity of nucleic acid sample, the method comprising thesteps of: obtaining a sample comprising nucleic acids; amplifying fromthe sample a plurality of first markers to provide a plurality of firstamplicons using at least one first oligonucleotide, wherein the firstoligonucleotide comprises a first dye; amplifying from the sample aplurality of second markers to provide a plurality of second ampliconsusing at least one second oligonucleotide, wherein the secondoligonucleotide comprises a second dye wherein, the first dye and thesecond dye are different dyes; and comparing a dye intensity level fromthe amplification of the plurality of first markers to a dye intensitylevel from the amplification of the plurality of second markers.

In certain aspects, the nucleic acids are DNA. In other aspects, thenucleic acids are cell free DNA. In one embodiment, the first dye isFAM. In another embodiment, the second dye is TET.

In certain aspects, the plurality of first amplicons are between about 1and 180 base pairs in length. In other aspects, the plurality of firstamplicons are between about 50 and about 70 based pairs in length. Inyet other aspects, the plurality of second amplicons are between about250 and 1,000 base pairs in length. In one embodiment, the plurality ofsecond amplicons are between about 400 and 500 base pairs in length.

In some embodiments, the plurality of first markers consists of between4 and 10 markers. In other embodiments, plurality of first markersconsists of between 5 and 7 markers. In yet other embodiments, theplurality of second markers consists of between 4 and 10 markers. In oneaspect, the plurality of second markers consists of between 4 and 7markers.

In some aspects, the method further comprises sequencing at least aportion of the nucleic acids in the sample. In one aspect, an amount ofthe sample sequenced is at least partially determined based on thecomparison of the dye intensity levels. In some embodiments, thesequencing comprises whole exome sequencing.

In other aspects, the method further comprises performing anamplification using at least one first oligonucleotide without thesample as a negative control. In one aspect, the method furthercomprises performing an amplification using at least one secondoligonucleotide without the sample as a negative control.

In some embodiments, the amplification steps occur during one or morePCR reactions. In one aspect, the PCR is digital PCR. In another aspect,the digital PCR is droplet digital PCR.

The present invention is also related to a method of selecting aplurality of first markers and a plurality of second markers fordetermining integrity of a nucleic acid sample, the method comprisingthe steps of: obtaining expression information on a genomic scale forpotential markers; selecting the plurality of first markers based on theexpression information, wherein the plurality of first markers areselected based on conditions in which the first markers are expressed;and selecting the plurality of second markers based on the expressioninformation, wherein the plurality of second markers are selected basedon conditions in which the second markers are expressed, wherein theplurality of first markers and the plurality of second markers do notoverlap.

In one embodiment, the expression information is obtained from at leastone of one or more databases, RNA sequencing analysis, microarrays, andreverse transcriptase PCR experiments.

In yet other aspects, the present invention is directed to a method ofdetermining integrity of nucleic acid sample, the method comprising thesteps of: obtaining a sample comprising nucleic acids; amplifying fromthe sample a plurality of first markers to provide a plurality of firstamplicons using at least one first oligonucleotide, wherein theplurality of first amplicons are between about 1 and 180 base pairs inlength; amplifying from the sample a plurality of second markers toprovide a plurality of second amplicons using at least one secondoligonucleotide, wherein the plurality of second amplicons are betweenabout 250 and 1,000 base pairs in length; and comparing a mass of theplurality of the first amplicons to a mass of the plurality of secondamplicons.

In certain aspects, the label on the oligonucleotide probe is afluorescent label and the label is selected from the group of FAM™ (5-or 6-carboxyfluorescein), Fluorescein, TET™ (5-tetrachloro-fluorescein),MARINA BLUE® (6,8-difluoro-7-hydroxy-4-methylcoumarin), ALEXA FLUOR® 350(7-amino-4-methyl-6-sulfocoumarin-3-acetic acid), YAKIMA YELLOW®(2′,5,5′,6-tetrachloro-7′-{12-[di(propan-2-yl)amino]-15-hydroxy-3-oxo-11,13-dioxa-4-aza-12-phosphapentadecyl}-4′-methyl-3-oxo-3H-spiro[2-benzofuran-1,9′-xanthene]-3′,6′-diylbis(2,2-dimethylpropanoate), and TEXAS RED® (sulforhodamine sulfonylchloride). Fluorescent labels (i.e., dyes) along with their channel fordetection and the excitation and detection wavelengths are provided inTable 1.

TABLE 1 Excitation source/Detection Channel filter Detected Dyes(Examples) Blue 365 ± 20 nm/460 ± 15 nm MARINA BLUE ®,(6,8-difluoro-7-hydroxy-4- methylcoumarin), ALEXA FLOUR ® 350(7-amino-4- methyl-6-sulfocoumarin-3- acetic acid) Green 470 ± 10 nm/510± 5 nm  FAM ™, (5- or 6- carboxyfluorescein), Fluorescein Yellow 530 ± 5nm/555 ± 5 nm TET ™ (5-tetrachloro- fluorescein), YAKIMA YELLOW ®(2′,5,5′,6- tetrachloro-7′-{12-[di(propan- 2-yl)amino]-15-hydroxy-3-oxo-11,13-dioxa-4-aza-12- phosphapentadecyl}-4′-methyl-3-oxo-3H-spiro[2-benzofuran- 1,9′-xanthane]-3′,6′-diylbis(2,2-dimethylpropanoate) Orange 585 ± 5 nm/610 ± 5 nm TEXAS RED ®(sulforhodamine sulfonyl chloride) Red 625 ± 10 nm/660 ± 10 nm Crimson 680 ± 5 nm/712 long pass

There is now strong evidence that the level of fetal cfDNA (and/or totalcfDNA) present in the circulatory system (e.g. in plasma) of a pregnantfemale is a marker of one or more forms of preeclampsia, such asearly-onset preeclampsia, mild and/or severe preeclampsia. The presentinvention shows particular utility in the efficient, effective,sensitive and/or low-variability detection/quantification of fetal cfDNApresent in plasma of pregnant females, and the present invention hasparticular utility therein. Accordingly, in particular embodiments ofthe present invention, the subject is a pregnant female and issusceptible to suffering or developing a pregnancy-associated medicalcondition; particularly where said pregnancy-associated medicalcondition is preeclampsia. As used herein, a subject “susceptible to” amedical condition may alternatively be described as “is suspected to” orto “be considered at risk of being susceptible to” suffering ordeveloping a medical condition; and in certain embodiments, the presentinvention is used to screen and/or diagnose the individual forsusceptibility to, risk of suffering or developing, or suffering from ordeveloping, a medical condition.

In alternative embodiments, the individual is a pregnant female and issusceptible to (or considered at risk of being susceptible to) sufferingor developing a pregnancy-associated medical condition selected from thegroup consisting of: preterm labor, intrauterine growth retardation andvanishing twin. The present invention may also be utilized in genderdetermination of twin pregnancies, by consideration of the relativevalues for fetal cfDNA compared to counts of Y-chromosome sequencesdetermined from cfDNA (e.g., by using parallel sequencing approaches).In these regards, it should be noted that approaches that usemassively-parallel sequencing of random cfDNA in maternal bloodtypically always count a very low frequency of “Y-chomomosone” sequences(such as between about 0.003% and 0.004% of all sequences, or betweenabout 0.0015% and 0.01% or 0.002% and 0.005% of all sequences) in allfemale pregnancies due to homology of certain Y-chromosome shortsequences to other chromosomes. A cut off “Y-chromosome” sequence countsof about 0.005%, or between about 0.003%, 0.004%, 0.006% or 0.007%, maytherefore be employed for female samples.

As described elsewhere herein, there is also increasing evidence thatthe presence and amount of certain forms of cfDNA is indicative orprognostic of certain medical conditions that are not associated withpregnancy. Accordingly, in another particular embodiment of the presentinvention, said species of DNA originates from a cell type associatedwith such a medical condition, particularly in those embodiments wheresaid species of DNA is circulating cell-free DNA and said sample is ablood fraction such as plasma or serum. For example, the medicalcondition may be a cell proliferative disorder, such as a tumor orcancer. In particular embodiments, the medical condition is a tumor or acancer of an organ selected from the list consisting of: liver, lung,breast, colon, esophagus, prostate, ovary, cervix, uterus, testis,brain, bone marrow and blood; and/or said species of DNA may originatefrom cells of a tumor; particularly where such tumor is a carcinoma orcancer of an organ selected from the group consisting of: liver, lung,breast, colon, esophagus, prostate, ovary, cervix, uterus, testis,brain, bone marrow and blood.

In yet another particular embodiment of the present invention, saidspecies of DNA originates from a cell type associated with a medicalcondition selected from the group consisting of: an infection/infectiousdisease, a wasting disorder, a degenerative disorder; an (auto)immunedisorder, kidney disease, liver disease, inflammatory disease, acutetoxicity, chronic toxicity, myocardial infarction, and a combination ofany of the forgoing (such as sepsis) and/or with a cell proliferativedisorder, particularly in those embodiments where said species of DNA iscirculating cell-free DNA and said sample is a blood fraction such asplasma or serum. For example, the medical condition may be aninfection/infectious disease, such as one caused by a bacterial, viralor protozoan pathogen, including a pathogen selected from the groupconsisting of: a retrovirus (such as HIV), a herpes virus (such as HSV,EBV, CMV, HHV or VSV), dengue virus, mycobacteria (e.g. Mycobacteriumtuberculosis), and hantavirus. In certain embodiments, the medicalcondition is sepsis and/or excludes kidney disease.

In some aspects of the present invention, there exist embodimentswherein the biological sample is a tissue sample or a sample ofbiological fluid. In particular, the sample is whole blood or a bloodfraction (e.g., such as plasma or serum). In alternative embodiments,the sample is biological fluid selected from the group consisting of:urine, saliva, sweat, ejaculate, teats, phlegm, vaginal secretion,vaginal wash and colonic wash. In more particular embodiments, thesample is a plasma or serum sample from the individual, or is urine fromthe individual in other embodiments, the sample is largely (oressentially) free from cells, and/or is not a whole blood and/orejaculate sample.

The present invention is further illustrated by the following examplesthat should not be construed as limiting. The contents of allreferences, patents, and published patent applications cited throughoutthis application, as well as the Figures, are incorporated herein byreference in their entirety for all purposes.

EXAMPLES Example 1. Materials and Methods

Control Genomic DNA

We obtained intact human genomic DNA samples from a commercial vendor(Sigma Aldrich). Control DNA from one individual was diluted to yield 4aliquots of 50 μL each a DNA concentration of 0.5 ng/μL. DNAconcentration was measured using QUBIT® dsDNA BR Fluorometric Assay(Life Technologies). We sheared each aliquot using sonication with E220™Focused-ultrasonicator (Covaris) as per manufacturer's instructions toachieve target fragment sizes were 150 bp, 300 bp, 500 bp and 1000 bp.The size distribution of DNA fragments was evaluated using theBioAnalyzer High Sensitivity Assay (Agilent Technologies, Inc.).

Control Plasma DNA

We obtained control plasma samples from healthy donors from a commercialvendor (BioreclamationIVT). Cell-free plasma DNA was extracted from 1 mLaliquots of plasma from 5 independently purchased samples using theQIAAMP® Circulating Nucleic Acid Kit (Qiagen). We modified therecommended protocol to elute in 100 μL volume and eluent was passedthrough the column twice. Plasma DNA was extracted with carrier RNAexcept when specifically indicated otherwise. Total DNA concentrationswere measured using the QUBIT® dsDNA BR Fluorometric Assay (LifeTechnologies). The size distribution of DNA fragments was evaluatedusing the BioAnalyzer High Sensitivity Assay (Agilent).

Assay Design

We designed a multiplexed assay targeting 9 single copy genomic lociexpected to be stable and least likely to be affected by copy numberevents in cancer patients. Five short PCR amplicons were designed withmean product size of ˜71 bp (range 67-75 bp) and all correspondingprobes were labeled with fluorescein amidite (FAM). Four long PCRamplicons were designed mean PCR product size of ˜471 bp (range 439-522bp) and all corresponding probes were labeled with5-tetrachloro-fluorescein (TET). Primers and probes were designed usingthe PRIMERQUEST® tool (IDT). Primers/probes were manually evaluated toensure they do not overlap with known polymorphic sites. In silico PCRwas used to confirm each primer yielded a single product and no crossproducts when used in multiplex. Primer and probe sequences are reportedin FIG. 1.

Droplet Digital PCR

A schematic presenting the steps involved in one application of digitaldroplet PCR to detect mutant and wild type genes in genomic DNA areshown in FIG. 2. Similarly, a multiplex ddPCR assay may be used todetect high molecular weight (HMW) DNA and low molecular weight (LMW)cfDNA in a biological sample containing both cfDNA and DNA derived fromcells (e.g., DNA from peripheral blood cells).

All digital droplet PCR reactions were prepared at 25 μL volume using12.50 μL of 2× Kapa Probe Fast Master Mix (Kapa Biosystems, USA), 1 μLof 5 mM dNTP Mix (Kapa), 1 μL of Droplet Stabilizer (RainDanceTechnologies, Lexington, Mass.), 1.25 μL of 20 uM of each primer (IDT)and 0.38 μL of 20 μM of each probe (IDT DNA, USA) and 2 μL of input DNA,and molecular biology grade water. Droplets were generated as permanufacturer's instructions using RAINDROP™ Digital PCR Source system(RainDance). Temperature cycling was performed using DNA ENGINE TETRAD®2 (Bio-Rad Laboratories, Hercules, Calif.) with the followingparameters: 1 cycle of 3 min at 95° C., 50 cycles of 15 sec at 95° C.and 1 min at 60° C. with a 0.5° C./sec ramp from 95° C. to 60° C., 1cycle of 98° C. for 10 min and hold at 4° C. forever. Dropletsfluorescence was measured using RAINDROP™ Digital PCR Sense system(RainDance).

Quantification of Plasma DNA

Analysis of fluorescence was performed using manufacturer's softwarethat accompanies RAINDROP™. Identification of positive droplets requiressetting fluorescence thresholds (gates) for each ddPCR assay. Wecompared results across intact genomic DNA and no template controls toassess thresholds for this assay. These thresholds were used for allfuture samples.

Based on the assay design, which is depicted in FIG. 3, we expect twopopulations of droplets on ddPCR, each representing the sum of productsfrom the two amplicon sets. Therefore, the number of FAM-positivedroplets (with the short PCR amplicon) represents 5 times the number ofamplifiable haploid copies of the genome (ACs) with fragment sizes >71bp (the average PCR amplicon size in the short set). This was used tocalculate total amplifiable plasma DNA concentration. Similarly, thenumber of TET-positive droplets (with the long PCR products) represents4 times the ACs with fragment sizes >471 bp (the average PCR ampliconsize in the long set). This was used to calculate concentration of HMWplasma DNA. The difference between the two sets was taken to be theconcentration of low molecular weight cfDNA. An example of thiscalculation is shown in FIG. 4. Quantification of fluorescence fromshort and large amplicons generated by the disclosed assay is depictedin FIG. 5.

Simulation of DNA Fragmentation and Expected Assay Performance

Simulations of DNA fragmentation were performed using R (code availableupon request). We assumed a DNA molecule spanning 2500 bp on either sideof the average amplicon size for each set. The length of this moleculewas 5071 bp for the short amplicon set and 5471 bp for the long ampliconset. This molecule was mathematically fragmented by sampling from anexponential distribution with a rate representing the targeted fragmentsize (150, 300, 500 or 1000 bp). We determined that a molecule will be“missed” by an amplicon if a DNA break fell within the amplicon region(bound by 5′ ends of either primers). We sampled 50,000 molecules todetermine overall frequency of missed molecules for each combination ofamplicon size and fragment size. The exponential distribution has beenused to model DNA shearing by sonication previously.

Exome Sequencing

Whole genome sequencing libraries from plasma and genomic DNA wereprepared using the THRUPLEX® DNA-Seq kit (Rubicon Genomics), as permanufacturer's instructions. Input cfDNA or sheared DNA was quantifiedusing the ddPCR assay described herein and diluted to 10 μL volume.Sample specific barcodes were assigned to each library. Each library wasquantified using the qPCR library quantification kits (Kapa). We pooledup to 8 libraries across 4 pools at equimolar concentrations inpreparation for exome enrichment. Exome enrichment was performed usingNimbleGen SeqCap EZ Human Exome v3 kit, as per manufacturer'sinstructions. We modified the protocol to use XGEN® Universal BlockingOligos—TS HT-i5 and TS HT-i7 (Integrated DNA Technologies).

All enriched exome libraries were quantified using qPCR and pooled atequimolar concentrations for sequencing. Sequencing was performed on theMiSeq® using the TruSeq® v3 150-cycle kit (Illumina) to generate 75 bppaired-end reads and 6 bp barcode read.

Sequencing Data Analysis

Raw data from the sequencer was demultiplexed and converted to fastqfiles using Picard tools. Demulitplexing on sample-specific barcodes wasperformed allowing zero mismatches and requiring minimum base qualityphred score of 30. Sequencing reads were aligned to the human genomehg19 using bwa v1.1. Aligned files were sorted and indexed usingsamtools v1.0. We estimated identified duplicate sequencing reads,estimated library complexity and estimated quality metrics (such ason-target reads) using Picard.

Example 2. Assessment of Assay Background

We evaluated the ddPCR assay using 3 template-free controls, 2 maize DNAsamples and 2 canine DNA samples to assess background noise. The maximumnumber of positive droplets observed in template-free controls (limit ofblank) was 28 and 20 for short and long amplicons respectively,equivalent to 6 and 5 ACs. The number of positive droplets across maizeand canine DNA was higher than template-free controls likely due tocross-amplification across species for conserved genomic regions.

Example 3. Evaluation of the Integrity and Quantification of ShearedGenomic DNA

The results of the ddPCR assessment of intact genomic DNA and fragmentedgenomic DNA are shown in FIG. 6 and FIG. 7. FIG. 6 presents a data setwith the raw fluorescence intensity measurements from ampliconsgenerated with fragmented DNA. FIG. 7 shows the genome equivalents permicroliter of sample for intact genomic DNA and fragmented genomic DNAhaving target fragment sizes of 1000 bp, 500 bp, 300 bp, and 150 bp ascalculated from the fluorescence intensity measurements.

The ddPCR assessment of intact genomic DNA at a QUBIT®-quantifiedconcentration of 0.5 ng/μL yielded 175 short and 183 long amplifiablecopies. The number of short ACs remained generally unaffected acrosssheared DNA samples except a significant drop with 150 bp fragments. Incontrast, the number of long ACs dropped with each decrease in averagefragment size (see FIG. 7).

The trend in long ACs was similar to that observed in simulated databased on exponential distributions with rates corresponding to averagefragment sizes. Surprisingly, simulated data underestimated the numberof short ACs for all fragment sizes demonstrating that the ddPCR assaytargeting short amplicons was more effective than estimated at producingACs. The actual and simulated curves are presented in FIG. 8.

Example 4. Assessment of Library Yield for Sheared vs. Intact DNA

Whole genome sequencing libraries were generated using intact DNA orsheared DNA comprising 25%, 50%, or 75% DNA fragments of about 300 bp.The input amounts of DNA used to generate the libraries were either 2.5ng or 5 ng. The library yield resulting from the various inputs wasdetermined by qPCR.

As shown in FIG. 9, intact DNA produced no detectable amount of librarysequences. In contrast, sheared DNA produced increasing yields oflibrary sequences as the relative percentage of DNA fragments of about300 bp increased. The greatest library yield resulted from 5 ng of DNAcontaining 75% fragments of about 300 bp.

These results indicate that intact DNA present in biological sampleswith cfDNA generally does not contribute to the sequencing libraryyield. Moreover, as the amount of low molecular weight cfDNA increasesin a biological sample the resulting yield of the library produced fromthis cfDNA will likewise increase.

Example 5. Preparation of cfDNA from Biological Samples with DifferingAmounts of HMW and LMW DNA

Preparation of cfDNA for whole genome sequencing assumes pre-fragmentedDNA, and no further shearing of the DNA is performed. However, resultsvary from one preparation of cfDNA to another. To investigate the causesfor this variation cfDNA was prepared from two biological samples andthe HMW DNA (i.e., large amplicons) and LMW cfDNA (i.e., smallamplicons) were quantified using the ddPCR method described in Example1.

Sample 1 generated a greater number of ACs than did Sample 2 usingsimilar amounts of starting material (compare the greater fluorescenceobserved with ddPCR of DNA from Sample 1 to that observed with ddPCR ofDNA from Sample 2 in FIG. 10A). When the relative amounts of largeamplicons and small amplicons produced from each sample were compared itwas seen that Sample 1 had a larger ratio of small amplicons to largeamplicons than Sample 2 (see FIG. 10B). These results suggest that theassessment of HMW and LMW DNA in biological samples using the methodsdisclosed herein provides an accurate indicator of the quality andquantity of cfDNA present in the samples.

Example 6. Diversity of ddPCR-Optimized Plasma Exome Libraries

ddPCR assessment of cfDNA in control plasma samples was used to guidelibrary preparation. Sequencing libraries were assessed for diversity(i.e., “estimated library diversity”) and duplication rate (i.e.,“number of genome equivalents”). Library diversity and size were relatedto input DNA as quantified using the ddPCR assay (see FIG. 11). As theamount of ddPCR optimized cfDNA used for library preparation increasedso did the library diversity and duplication rate.

Example 7. Assessment of the Effects of Upstream Processing ofBiological Samples on cfDNA Amplifiable Copies

Prior to analysis of cfDNA with whole genome sequencing several upstreamprocesses are required to collect and process biological samples. Theseupstream processes include such things as collection of biologicalsamples (e.g., blood, urine, saliva) and extraction of DNA from thebiological samples. The upstream processes can affect the downstreamanalysis and sequencing of cfDNA.

The ddPCR assay described in Example 1 was used to evaluate the effectof various blood collection protocols. The protocols employed bloodcollection tubes containing EDTA as the anticoagulant or Streck cfDNAblood collection tubes (Streck Laboratories, Omaha, Nebr.) containing aproprietary preservative (Streck Laboratories). The effect of thevarious blood collection protocols on the number of cfDNA amplifiablecopies per mL of plasma is shown in FIG. 12.

DNA may be extracted from biological samples in several ways, and thisupstream process also affects the downstream analysis and sequencing ofcfDNA. Several DNA extraction kits using spin columns or magnetic beadswere evaluated for their effect on the cfDNA in plasma samples. The DNAin each sample was extracted per the manufacturer's instructions in eachkit. cfDNA amplifiable copies per mL plasma in each biological samplewere determined with the ddPCR assay described in Example 1. The resultsof the analysis presented in FIGS. 13A and 13B show that the extractionkit used can significantly impact the amount of cfDNA amplifiable copiesobtained from plasma samples.

The ddPCR assay described herein can also be used to analyze the qualityof biological samples from different study cohorts. Such an analysis wasperformed resulting in the data shown in FIG. 14. This qualityassessment across study cohorts can be useful in interpreting theresults of the data and explaining differences observed across thecohorts. For example, if one cohort has a relatively high level of cfDNAamplifiable copies in its biological samples the subsequent whole genomesequencing and analysis may identify genetic indicators of disease thatare not detectable in other cohorts due the poor quality or relativelylow levels of cfDNA amplifiable copies in their biological samples.

The experimental data outlined above demonstrate that the multiplexedddPCR assay allows accurate and precise assessment of amplifiable copiesand integrity of cfDNA from small amounts of input (e.g., picograms ofDNA). Upfront quality assessment with the multiplexed ddPCR assay canoptimize downstream analysis and sequencing. Moreover, the multiplexedddPCR assay provides reliable metrics for optimization of pre-analyticalfactors such as plasma processing and extraction.

Unless defined otherwise, all technical and scientific terms herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materials,similar or equivalent to those described herein, can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein. All publications, patents, and patentpublications cited are incorporated by reference herein in theirentirety for all purposes.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

REFERENCES

-   1. Bianchi D W, Parker R L, Wentworth J, Madankumar R, Saffer C, Das    A F, et al. DNA sequencing versus standard prenatal aneuploidy    screening. The New England journal of medicine 2014; 370:799-808.-   2. Leary R J, Sausen M, Kinde I, Papadopoulos N, Carpten J D, Craig    D, et al. Detection of chromosomal alterations in the circulation of    cancer patients with whole-genome sequencing. Science translational    medicine 2012; 4:162ra54.-   3. Chan K C, Jiang P, Zheng Y W, Liao G J, Sun H, Wong J, et al.    Cancer genome scanning in plasma: Detection of tumor-associated copy    number aberrations, single-nucleotide variants, and tumoral    heterogeneity by massively parallel sequencing. Clinical chemistry    2013; 59:211-24.-   4. Murtaza M, Dawson S J, Tsui D W, Gale D, Forshew T, Piskorz A M,    et al. Non-invasive analysis of acquired resistance to cancer    therapy by sequencing of plasma DNA. Nature 2013; 497:108-12.-   5. Chiu R W, Akolekar R, Zheng Y W, Leung T Y, Sun H, Chan K C, et    al. Non-invasive prenatal assessment of trisomy 21 by multiplexed    maternal plasma DNA sequencing: Large scale validity study. Bmj    2011; 342:c7401.-   6. Lo Y M, Chan K C, Sun H, Chen E Z, Jiang P, Lun F M, et al.    Maternal plasma DNA sequencing reveals the genome-wide genetic and    mutational profile of the fetus. Sci Transl Med 2010; 2:61ra91.-   7. De Vlaminck I, Valantine H A, Snyder™, Strehl C, Cohen G, Luikart    H, et al. Circulating cell-free DNA enables noninvasive diagnosis of    heart transplant rejection. Sci Transl Med 2014; 6:241ra77.-   8. Forshew T, Murtaza M, Parkinson C, Gale D, Tsui D W, Kaper F, et    al. Noninvasive identification and monitoring of cancer mutations by    targeted deep sequencing of plasma DNA. Science translational    medicine 2012; 4:136ra68.-   9. Dawson S J, Tsui D W, Murtaza M, Biggs H, Rueda O M, Chin S F, et    al. Analysis of circulating tumor DNA to monitor metastatic breast    cancer. The New England journal of medicine 2013; 368:1199-209.-   10. Bettegowda C, Sausen M, Leary R J, Kinde I, Wang Y, Agrawal N,    et al. Detection of circulating tumor DNA in early- and late-stage    human malignancies. Science translational medicine 2014; 6:224ra24.-   11. Devonshire A S, Whale A S, Gutteridge A, et al. Towards    standardisation of cell-free DNA measurement in plasma: controls for    extraction efficiency, fragment size bias and quantification.    Analytical and Bioanalytical Chemistry. 2014; 406(26):6499-6512.

What is claimed is:
 1. A method of determining and optimizing theintegrity of cell free DNA (cfDNA) in a biological sample for librarypreparation, the method comprising the steps of: a) obtaining abiological sample comprising cfDNA; b) contacting the biological samplewith at least a first primer/probe set and at least a second primerprobe/set, each of which comprises at least one oligonucleotide probethat is detectably labeled and at least two oligonucleotide primers andthe labels are different for each primer/probe set, under conditionssuch that the first primer/probe set anneals to a first target sequenceof less than 300 bp in length and the second primer/probe set anneals toa second target sequence of 300 bp or greater in length; c) amplifying,if present, the first target sequence and the second target sequence;and d) monitoring for detection of the label of the oligonucleotideprobe from the first primer/probe set as an indication of hybridizationto the first target sequence and detection of the label of theoligonucleotide probe from the second primer/probe set as an indicationof hybridization to the second target sequence; e) determining theintegrity of the cfDNA by calculating the ratio of the level ofdetection of the label of the oligonucleotide probe from the firstprimer/probe set to the level of detection of the label of theoligonucleotide probe from the second primer/probe set, wherein agreater ratio indicates increased integrity of cfDNA in the biologicalsample; and f) generating a sequencing library with the cfDNA based onthe optimized integrity of the cfDNA, wherein an increase in the amountof optimized cfDNA used for library preparation increases the librarydiversity and duplication rate.
 2. The method of claim 1, wherein theoligonucleotide primers have a nucleotide sequence length of about 10 toabout
 150. 3. The method of claim 1, wherein the oligonucleotide probeshave a nucleotide sequence length of about 10 to about
 50. 4. The methodof claim 1, wherein the first target sequence and the second targetsequence are from at least one housekeeping gene.
 5. The method of claim4, wherein the at least one housekeeping gene is selected from the groupconsisting of ACTB (Beta-actin), GAPDH (Glyceraldehyde 3-phosphatedehydrogenase), RPLP0 (60 S acidic ribosomal protein P0), GUSB(beta-glucuronidase), and TFRC (transferring receptor 1).
 6. The methodof claim 1, wherein the first primer/probe set comprises at least oneoligonucleotide probe and at least two oligonucleotide primers eachcomprising an oligonucleotide sequence selected from the groupconsisting of SEQ ID NOs: 13-27.
 7. The method of claim 1, wherein thesecond primer/probe set comprises at least one oligonucleotide probe andat least two oligonucleotide primers each comprising an oligonucleotidesequence selected from the group consisting of SEQ ID NOs: 1-12.
 8. Themethod of claim 1, wherein the first primer/probe set comprises at leasttwo, at least three, at least four, or at least five oligonucleotideprobes that are detectably labeled and at least four, at least six, atleast eight, or at least 10 oligonucleotide primers, each of whichanneals to a target sequence of less than 300 bp, and wherein detectionof the labels of the oligonucleotide probes from the first primerprobe/set are averaged to determine the detection of the labels.
 9. Themethod of claim 1, wherein the second primer/probe set comprises atleast two, at least three, at least four, or at least fiveoligonucleotide probes that are detectably labeled and at least four, atleast six, at least eight, or at least 10 oligonucleotide primers, eachof which anneals to a target sequence of 300 bp or greater in length,and wherein detection of the labels of the oligonucleotide probes fromthe second primer probe/set are averaged to determine the detection ofthe labels.
 10. The method of claim 1, wherein the amplifying of thetarget sequences occurs with a digital PCR technique selected fromdroplet digital PCR (ddPCR), BEAMing (beads, emulsion, amplification,and magnetic), and microfluidic chips.
 11. The method of claim 10,wherein the digital PCR technique is multiplexed ddPCR, and amplifiablecopies (ACs) of cfDNA in the biological sample are estimated by: (i)dividing the number of positive droplets indicating hybridization to thefirst target sequence by the number of oligonucleotide probes in thefirst primer/probe set; (ii) dividing the number of positive dropletsindicating hybridization to the second target sequence by the number ofoligonucleotide probes in the second primer/probe set; and (iii)subtracting (ii) from (i) to estimate ACs of cfDNA in the biologicalsample.
 12. The method of claim 1, wherein the oligonucleotide probesare detectably labeled with a fluorescent label selected from the groupconsisting of 5 or 6-carboxyfluorescein, Fluorescein,5-tetrachloro-fluorescein, sulforhodamine sulfonyl chloride,2′,5,5′,6-tetrachloro-7′-{12-[di(propan-2-yl)amino]-15-hydroxy-3-oxo-11,13-dioxa-4-aza-12-phosphapentadecyl}-4′-methyl-3-oxo-3H-spiro[2-benzofuran-1,9′-xanthene]-3′,6′-diylbis(2,2-dimethylpropanoate, and 6,8-difluoro-7-hydroxy-4-methylcoumarin.13. The method of claim 12, wherein the fluorescent labels are 5- or6-carboxyfluorescein and 5-tetrachloro-fluorescein.
 14. The method ofclaim 1, wherein the first primer/probe set detects at least one, atleast two, at least three, at least four, or at least five targetsequences of about 50 bp to about 100 bp in length.
 15. The method ofclaim 1, wherein the second primer/probe set detects at least one, atleast two, at least three, at least four, or at least five targetsequences of about 300 bp to about 1000 bp in length.
 16. The method ofclaim 1, wherein the sequencing library is an exome library.
 17. Themethod of claim 1, wherein the biological sample is divided intoaliquots and each aliquot undergoes a different upstream process toevaluate how upstream processing affects the integrity of cfDNA in thebiological sample.
 18. The method of claim 1, wherein the biologicalsample is a biofluid selected from the group consisting of blood,plasma, serum, saliva, urine, tears, and cerebral spinal fluid.
 19. Themethod of claim 1, wherein detection of the labels of theoligonucleotide probes occurs with a flow cytometer or a droplet reader.